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- EMDB-11985: TRPC4 in complex with Calmodulin -

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Basic information

Entry
Database: EMDB / ID: EMD-11985
TitleTRPC4 in complex with Calmodulin
Map data
Sample
  • Organelle or cellular component: TRPC4 membrane protein in complex with calmodulin
    • Organelle or cellular component: Transient receptor potential cation channel subfamily c member 4a
      • Protein or peptide: Transient receptor potential cation channel subfamily c member 4a
    • Organelle or cellular component: Calmodulin-1
      • Protein or peptide: Calmodulin-1
  • Ligand: CALCIUM IONCalcium
  • Ligand: (2R)-3-(phosphonooxy)propane-1,2-diyl dihexanoate
Function / homology
Function and homology information


CaMK IV-mediated phosphorylation of CREB / Cam-PDE 1 activation / CREB1 phosphorylation through the activation of CaMKII/CaMKK/CaMKIV cascasde / Glycogen breakdown (glycogenolysis) / Activation of RAC1 downstream of NMDARs / Reduction of cytosolic Ca++ levels / Sodium/Calcium exchangers / Activation of Ca-permeable Kainate Receptor / CLEC7A (Dectin-1) induces NFAT activation / Synthesis of IP3 and IP4 in the cytosol ...CaMK IV-mediated phosphorylation of CREB / Cam-PDE 1 activation / CREB1 phosphorylation through the activation of CaMKII/CaMKK/CaMKIV cascasde / Glycogen breakdown (glycogenolysis) / Activation of RAC1 downstream of NMDARs / Reduction of cytosolic Ca++ levels / Sodium/Calcium exchangers / Activation of Ca-permeable Kainate Receptor / CLEC7A (Dectin-1) induces NFAT activation / Synthesis of IP3 and IP4 in the cytosol / RHO GTPases activate PAKs / Calmodulin induced events / Inactivation, recovery and regulation of the phototransduction cascade / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation / Calcineurin activates NFAT / eNOS activation / Ion transport by P-type ATPases / Unblocking of NMDA receptors, glutamate binding and activation / Protein methylation / RAF activation / VEGFR2 mediated vascular permeability / RAS processing / Smooth Muscle Contraction / Ca2+ pathway / FCERI mediated Ca+2 mobilization / RAF/MAP kinase cascade / RHO GTPases activate IQGAPs / Extra-nuclear estrogen signaling / store-operated calcium channel activity / PKA activation / Platelet degranulation / cation channel complex / Stimuli-sensing channels / Ion homeostasis / type 3 metabotropic glutamate receptor binding / inositol 1,4,5 trisphosphate binding / organelle localization by membrane tethering / regulation of cardiac muscle cell action potential / mitochondrion-endoplasmic reticulum membrane tethering / autophagosome membrane docking / nitric-oxide synthase binding / protein phosphatase activator activity / monoatomic cation transport / adenylate cyclase binding / catalytic complex / detection of calcium ion / negative regulation of ryanodine-sensitive calcium-release channel activity / regulation of cardiac muscle contraction / calcium channel inhibitor activity / cellular response to interferon-beta / voltage-gated potassium channel complex / phosphatidylinositol 3-kinase binding / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / monoatomic cation channel activity / positive regulation of protein dephosphorylation / titin binding / regulation of ryanodine-sensitive calcium-release channel activity / sperm midpiece / calcium channel complex / regulation of cytosolic calcium ion concentration / adenylate cyclase activator activity / regulation of heart rate / nitric-oxide synthase regulator activity / protein serine/threonine kinase activator activity / sarcomere / regulation of cytokinesis / spindle microtubule / calcium ion transmembrane transport / positive regulation of receptor signaling pathway via JAK-STAT / spindle pole / cellular response to type II interferon / response to calcium ion / calcium-dependent protein binding / G2/M transition of mitotic cell cycle / myelin sheath / growth cone / transmembrane transporter binding / protein domain specific binding / centrosome / calcium ion binding / protein kinase binding / protein-containing complex / nucleoplasm / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Transient receptor ion channel II / Transient receptor ion channel domain / Transient receptor ion channel II / Transient receptor potential channel, canonical / Ankyrin repeat / EF-hand domain pair / Ankyrin repeats (3 copies) / Ankyrin repeat profile. / ankyrin repeats / Ankyrin repeat ...Transient receptor ion channel II / Transient receptor ion channel domain / Transient receptor ion channel II / Transient receptor potential channel, canonical / Ankyrin repeat / EF-hand domain pair / Ankyrin repeats (3 copies) / Ankyrin repeat profile. / ankyrin repeats / Ankyrin repeat / Ankyrin repeat-containing domain superfamily / EF-hand, calcium binding motif / EF-Hand 1, calcium-binding site / EF-hand calcium-binding domain. / EF-hand calcium-binding domain profile. / Ion transport domain / Ion transport protein / EF-hand domain / EF-hand domain pair
Similarity search - Domain/homology
Calmodulin-1 / Transient receptor potential cation channel subfamily c member 4a
Similarity search - Component
Biological speciesDanio rerio (zebrafish) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsVinayagam D / Quentin D / Sistel O / Merino F / Stabrin M / Hofnagel O / Ledeboer MW / Malojcic G / Raunser S
Funding support Germany, 1 items
OrganizationGrant numberCountry
Max Planck Society Germany
CitationJournal: Elife / Year: 2020
Title: Structural basis of TRPC4 regulation by calmodulin and pharmacological agents.
Authors: Deivanayagabarathy Vinayagam / Dennis Quentin / Jing Yu-Strzelczyk / Oleg Sitsel / Felipe Merino / Markus Stabrin / Oliver Hofnagel / Maolin Yu / Mark W Ledeboer / Georg Nagel / Goran ...Authors: Deivanayagabarathy Vinayagam / Dennis Quentin / Jing Yu-Strzelczyk / Oleg Sitsel / Felipe Merino / Markus Stabrin / Oliver Hofnagel / Maolin Yu / Mark W Ledeboer / Georg Nagel / Goran Malojcic / Stefan Raunser /
Abstract: Canonical transient receptor potential channels (TRPC) are involved in receptor-operated and/or store-operated Ca signaling. Inhibition of TRPCs by small molecules was shown to be promising in ...Canonical transient receptor potential channels (TRPC) are involved in receptor-operated and/or store-operated Ca signaling. Inhibition of TRPCs by small molecules was shown to be promising in treating renal diseases. In cells, the channels are regulated by calmodulin (CaM). Molecular details of both CaM and drug binding have remained elusive so far. Here, we report structures of TRPC4 in complex with three pyridazinone-based inhibitors and CaM. The structures reveal that all the inhibitors bind to the same cavity of the voltage-sensing-like domain and allow us to describe how structural changes from the ligand-binding site can be transmitted to the central ion-conducting pore of TRPC4. CaM binds to the rib helix of TRPC4, which results in the ordering of a previously disordered region, fixing the channel in its closed conformation. This represents a novel CaM-induced regulatory mechanism of canonical TRP channels.
History
DepositionNov 24, 2020-
Header (metadata) releaseDec 9, 2020-
Map releaseDec 9, 2020-
UpdateDec 9, 2020-
Current statusDec 9, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0052
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0052
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7b1g
  • Surface level: 0.0052
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11985.png

Downloads & links

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Map

FileDownload / File: emd_11985.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.85 Å
Density
Contour LevelBy AUTHOR: 0.0052 / Movie #1: 0.0052
Minimum - Maximum-0.013438393 - 0.023819605
Average (Standard dev.)0.00013845753 (±0.0009158334)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 244.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.850.850.85
M x/y/z288288288
origin x/y/z0.0000.0000.000
length x/y/z244.800244.800244.800
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ240240240
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS288288288
D min/max/mean-0.0130.0240.000

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Supplemental data

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Sample components

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Entire : TRPC4 membrane protein in complex with calmodulin

EntireName: TRPC4 membrane protein in complex with calmodulin
Components
  • Organelle or cellular component: TRPC4 membrane protein in complex with calmodulin
    • Organelle or cellular component: Transient receptor potential cation channel subfamily c member 4a
      • Protein or peptide: Transient receptor potential cation channel subfamily c member 4a
    • Organelle or cellular component: Calmodulin-1
      • Protein or peptide: Calmodulin-1
  • Ligand: CALCIUM IONCalcium
  • Ligand: (2R)-3-(phosphonooxy)propane-1,2-diyl dihexanoate

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Supramolecule #1: TRPC4 membrane protein in complex with calmodulin

SupramoleculeName: TRPC4 membrane protein in complex with calmodulin / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 / Details: TRPC4 was solubilised in LMNG detergent
Molecular weightTheoretical: 126 KDa

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Supramolecule #2: Transient receptor potential cation channel subfamily c member 4a

SupramoleculeName: Transient receptor potential cation channel subfamily c member 4a
type: organelle_or_cellular_component / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Danio rerio (zebrafish) / Location in cell: plasma membrane
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK 293 GnTI- / Recombinant plasmid: pEG BACMAM

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Supramolecule #3: Calmodulin-1

SupramoleculeName: Calmodulin-1 / type: organelle_or_cellular_component / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)

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Macromolecule #1: Transient receptor potential cation channel subfamily c member 4a

MacromoleculeName: Transient receptor potential cation channel subfamily c member 4a
type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Danio rerio (zebrafish)
Molecular weightTheoretical: 105.204641 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GSQLYFRRTD NSSYRDRIPL RIVRAESELS TQEKSYLSAV EKGDYASVKL ALEEAEIYFK ININCIDPLG RTALLIAIEN ENLEIIELL LSFNVYVGDA LLHAIRKEVV GAVELLLNHK KPSGEKQVPP ILLDKQFSDF TPDITPIILA AHTNNYEIIK M LVQKGVSV ...String:
GSQLYFRRTD NSSYRDRIPL RIVRAESELS TQEKSYLSAV EKGDYASVKL ALEEAEIYFK ININCIDPLG RTALLIAIEN ENLEIIELL LSFNVYVGDA LLHAIRKEVV GAVELLLNHK KPSGEKQVPP ILLDKQFSDF TPDITPIILA AHTNNYEIIK M LVQKGVSV PQPHEVRCNC VECVSSSDVD SLRHSRSRLN IYKALASPSL IALSSEDPFL TAFQLSWELQ ELSKVENEFK AE YEELSHQ CKHFAKDLLD QTRSSRELEL ILNFRDDMNL LQDEANNELA RLKLAIKYRQ KEFVAQPNCQ QLLASRWYDE FPG WRRRHW AGKLITCVFI GLMFPLLSLC YLVAPKSRYG LFIRKPFIKF ICHTASYLTF LFLLLLASQH IVSNNPDRQG PKPT TVEWM ILPWVLGFIW TEIKQMWDGG FQDYIHDWWN LMDFVMNSLY LATISLKIVA YVKYSGCKPR DTWEMWHPTL VAEAV FAIA NIFSSLRLIS LFTANSHLGP LQISLGRMLL DILKFLFIYC LVLLAFANGL NQLYFYYENS EGMTCKGIRC ERQNNA FST LFETLQSLFW SIFGLISLYV TNVKADHKFT EFVGATMFGT YNVISLVVLL NMLIAMMNNS YQHIADHADI EWKFART KL WMSYFEEGGT LPPPFNIIPS PKSICYLITW IKVHVFKRRS KRTETFGTLG RRAAENVRLN HQYQEVLRNL VKRYVAAM I RDAKTEEGLT EENFKELKQD ISSFRYEVIG MMKGNRKSTR ANKSDTSASD VSHPEGSLQY SSALKQNSKL HLYDVTTAL QQQNSEEAKA SLGCLANGSA VVLTEPILKD KARSDFPKDF TDFGLFPKKQ NPNKIYSLAE EATESDPDIL DWGKEDKPLA GKVEQDVNE SKCLMEEDER VLEEQEMEHI ASSHEH

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Macromolecule #2: Calmodulin-1

MacromoleculeName: Calmodulin-1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 16.852545 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString:
MADQLTEEQI AEFKEAFSLF DKDGDGTITT KELGTVMRSL GQNPTEAELQ DMINEVDADG NGTIDFPEFL TMMARKMKDT DSEEEIREA FRVFDKDGNG YISAAELRHV MTNLGEKLTD EEVDEMIREA DIDGDGQVNY EEFVQMMTAK

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Macromolecule #3: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 3 / Number of copies: 4 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #4: (2R)-3-(phosphonooxy)propane-1,2-diyl dihexanoate

MacromoleculeName: (2R)-3-(phosphonooxy)propane-1,2-diyl dihexanoate / type: ligand / ID: 4 / Number of copies: 4 / Formula: 44E
Molecular weightTheoretical: 368.36 Da
Chemical component information

ChemComp-44E:
(2R)-3-(phosphonooxy)propane-1,2-diyl dihexanoate / phospholipid*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.35 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3TRIS
150.0 mMNaClSodium chloridesodium chloride
0.003 percentC47H88O26LMNG
GridModel: Quantifoil R2/1 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated magnification: 59000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.001 mm / Nominal defocus max: 2.8 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 130000
Specialist opticsSpherical aberration corrector: Cs corrector first generation
Energy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 160.0 K / Max: 190.0 K
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-80 / Number grids imaged: 1 / Number real images: 7972 / Average exposure time: 10.0 sec. / Average electron dose: 88.52 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 678331
CTF correctionSoftware - Name: CTFFIND (ver. 4.1.10)
Startup modelType of model: EMDB MAP
EMDB ID:

4339

Initial angle assignmentType: NOT APPLICABLE / Details: Used EMD-4339 as a reference
Final 3D classificationNumber classes: 10 / Software - Name: SPHIRE (ver. 1.3) / Software - details: SORT3D
Details: Symmetry expansion was used to quadruple the Particles. From the best classes after 3d classification duplicates were removed that resulted in 160829 particles
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: SPHIRE / Software - details: MERIDIEN
Final reconstructionNumber classes used: 4 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: SPHIRE / Software - details: MERIDIEN / Number images used: 160829

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Atomic model buiding 1

Initial model(PDB ID:

6gik

,

2lv6

)
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 58.04 / Target criteria: Correlation coefficient
Output model

PDB-7b1g:
TRPC4 in complex with Calmodulin

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